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Invited Review

Visceral leishmaniasis treatment: What do we have, what do we need and how


to deliver it?
Lucio H. Freitas-Junior
a,
, Eric Chatelain
b
, Helena Andrade Kim
a,1
, Jair L. Siqueira-Neto
a
a
Center for Neglected Diseases Drug Discovery (CND3), Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea
b
Drugs for Neglected Diseases initiative (DNDi), 15 Chemin Louis Dunant, Geneva 1202, Switzerland
a r t i c l e i n f o
Article history:
Received 26 September 2011
Received in revised form 12 January 2012
Accepted 14 January 2012
Available online 28 January 2012
Keywords:
Leishmania
Leishmaniasis
Drug discovery
High throughput screening
a b s t r a c t
Leishmaniasis is one of the most neglected tropical disease in terms of drug discovery and development.
Most antileishmanial drugs are highly toxic, present resistance issues or require hospitalization, being
therefore not adequate to the eld. Recently improvements have been achieved by combination therapy,
reducing the time and cost of treatment. Nonetheless, new drugs are still urgently needed.
In this review, we describe the current visceral leishmaniasis (VL) treatments and their limitations. We
also discuss the new strategies in the drug discovery eld including the development and implementa-
tion of high-throughput screening (HTS) assays and the joint efforts of international teams to deliver
clinical candidates.
2012 Australian Society for Parasitology Published by Elsevier Ltd. All rights reserved.
Contents
1. Leishmaniasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2. Current therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3. VL drug discovery: new treatments on the way . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
4. Compound screening: rapidly assessing hits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
5. The target-free screening approach. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
6. Hit-to-lead & lead optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
1. Leishmaniasis
Leishmaniasis is a group of diseases caused by trypanosomatids
from the genus Leishmania. Transmission occurs in 88 tropical and
subtropical countries where the sandy vector is present, meaning
that approximately 350 million people are at risk of contracting the
disease (WHO, 1990; Piscopo and Mallia Azzopardi, 2007). It is one
of the most neglected tropical diseases, with a major impact
among the poorest.
Leishmania parasites live a dual-form life cycle (digenetic life
cycle), as either a promastigote agellar or an amastigote form.
The promastigotes are found in the insect vector and are injected
into the mammalian host during the vectors blood meal. Then, they
are phagocytised by macrophages, dendritic cells and/or neutro-
phils attracted to the biting site in the skin. Once inside the phago-
some, promastigotes differentiate into amastigotes, multiply by
simple division until bursting the host cell. In the mammalian host,
these protozoa are obligate intracellular parasites of macrophage-
dendritic cell lineages. This complex life cycle includes several
facets that might be exploited for drug design optimization and
development (Hammarton et al., 2003). The parasites can have dif-
ferent host cells and organs tropism, infecting either supercial cells
or visceral cells, which results in hepatosplenomegaly and bone
2211-3207/$ - see front matter 2012 Australian Society for Parasitology Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijpddr.2012.01.003

Corresponding author. Tel.: +82 31 8018 8007.


E-mail address: freitasjunior@ip-korea.org (L.H. Freitas-Junior).
1
Present address: Department of Biomedical Engineering, Dongguk University, 3-
26 Phil-Dong, Joong-Gu, Seoul 100-715, South Korea.
International Journal for Parasitology: Drugs and Drug Resistance 2 (2012) 1119
Contents lists available at SciVerse ScienceDirect
International Journal for Parasitology:
Drugs and Drug Resistance
j our nal homepage: www. el sevi er . com/ l ocat e/ i j pddr
marrow inltration. Depending on the tropism, the disease can be
characterized by at least four syndromes: cutaneous leishmaniasis
(CL), muco-cutaneous leishmaniasis (MCL), visceral leishmaniasis
(VL) also known as kala-azar in the Indian sub continent or black
fever, which is the most severe form of the disease being fatal if un-
treated, and post-kala-azar dermal leishmaniasis (PKDL). Factors
determining the kind of clinical manifestation depend upon the
infecting species and host factors, such as general health, genetic
and immune constitution (Locksley et al., 1999; Tripathi et al.,
2007).
In non-endemic areas of the world, VL is mostly an opportunis-
tic infection with up to 70% of adult leishmaniasis cases being re-
lated to human immunodeciency virus (HIV) infection. The
greatest prevalence of reported co-infection in non-endemic areas
is in the Mediterranean area. Atypical presentations of VL are
reported in HIV patients, and HIV/AIDS clinical development is pro-
moted by VL (WHO, 2007).
The numbers of leishmaniasis cases are increasing worldwide.
Some reasons are the lack of vaccines, difculties in controlling
vectors and the increasing number of parasites resistance to
chemotherapy.
In the following sections we briey review the currently avail-
able chemotherapy and discuss the recent developments on the
drug discovery eld of VL.
2. Current therapies
Over the past decades, few alternative drugs or new formula-
tions of old ones have become available but, as yet, none of them
are ideal for treatment due to high toxicity, resistance issues, pro-
hibitive prices, long treatment length or inadequate mode of
administration not adapted to the eld (see Table 1). In addition,
many patients are unable to complete the whole treatment,
increasing the risk of drug resistance development. Drug combina-
tions have demonstrated positive results and may be a short-term
solution to delay or prevent the emergence of resistance, increas-
ing efcacy, or shortening the course of treatment (Alvar et al.,
2006; Sundar et al., 2011).
For many years, pentavalent antimonials have been the recom-
mended drug for VL and CL. Pentavalent antimonials meglumine
antimoniate (Glucantime, Sano-Aventis) and sodium stibogluco-
nate (Pentostan, GlaxoSmithKline) have variable efcacy against
VL andCL, andrequire injectableadministration, that canbe intrave-
nous (IV), intramuscular (IM) or intralymphatic (IL). Due to side
effects such as high cardiotoxicity (Matoussi et al., 2007), pancreati-
tis (Gasser et al., 1994; Shahian and Alborzi, 2009) and nephrotoxi-
city (Zaghloul and Al-Jasser, 2004), patients should be hospitalized
and monitored, as treatment may need to be suspended. There are
currently efforts to reduce toxicity and improve delivery of
antimonials (Frezard et al., 2009), with attempts including lipo-
some-based formulations for VL treatment (Schettini et al., 2006)
and cyclodextrin-based formulation for oral delivery (Demicheli
et al., 2004).
Antimonials seem to have a broad mechanism of action. Data
suggest pentavalent antimony (SbV) enters the host cells, crosses
the phagolysosomal membrane and is converted into trivalent
antimony (SbIII). Then, SbIII acts against amastigotes by compro-
mising the cells thiol redox potential by inducing efux of intracel-
lular thiols and consequently inhibiting trypanothione reductase
(TR) (Wyllie et al., 2004). SbV reduction can be non-enzymatic, un-
der acidic conditions such as those found in the phagolysosome, by
glutathione (GSH), glycylcysteine and trypanothione, or enzymatic
by thiol-dependent reductase (TDR1) (Denton et al., 2004) and
antimonite reductase (ACR2) (Ashutosh et al., 2007). ACR2 also in-
creases the sensitivity of Leishmania to SbV (Zhou et al., 2004).
SbVmay also kill parasites by indirect mechanisms, such as increas-
ing cytokine levels (Pathak and Yi, 2001). Antimonials also act at the
DNA level, inducing DNA damage in vivo (Lima et al., 2010), and
inhibiting DNAtopoisomerase I (Chakraborty and Majumder, 1988).
Amphotericin B deoxycholate (Fungizone) is a systemic anti-
fungal and a highly active antileishmanial. Due to the increasing
resistance to antimonials, it is used as an alternative drug for VL.
It is highly toxic, requiring careful and slow IV administration.
Amphotericin B complexes with 24-substituted sterols from the
biological membrane, such as ergosterol. These complexes open
pores which alter the ion balance and lead to cell death (Roberts
et al., 2003).
Lipid formulations of amphotericin B have been developed in
order to improve its bioavailability and pharmacokinetic (PK)
properties, considerably reducing side effects (Gangneux et al.,
1996; Yardley and Croft, 1997; Torchilin, 2005). The larger lipid
particles are rapidly assimilated by the mononuclear phagocyte
system (hepatic macrophages), where Leishmania donovani para-
sites accumulate and VL develops. Another advantage is that smal-
ler liposomes stay in the blood stream longer than the free drug
(Yardley and Croft, 1997). The liposomal formulation (AmBisome)
is an approved treatment for VL that besides the reduced toxicity
has a better half-life and a high level of efcacy, with 90% cure rate.
In experimental VL models, AmBisome has hepatic accumulation,
reaches therapeutic levels faster than antimonials and has a longer
half-life (Yardley and Croft, 1997; Berman et al., 1998; Sundar
et al., 2004). The main limitations are its high cost, administration
route and lack of stability at high temperature (cold chain is
needed).
Table 1
Current VL treatments and their main characteristics.
Drugs Administration Regimen Efcacy (
*
) Resistance Toxicity Price
Pentavalent
antimonials
IV, IM and IL 30 days
20 mg/kg/day
3595% (depending on
area)
Common (>60% in
Bihar, India)
+++ Cardiotoxicity, pancreatitis, nephrotoxicity,
hepatotoxicity
$50
70
Amphotericin B IV 30 days
1 mg/kg (15 mg/kg
total dose)
>90% Laboratory strains +++ Nephrotoxicity $100
Liposomal
amphotericin B
IV 520 mg/kg total dose
410 doses over 10
20 days
>97% Not documented +/ Rigors and chills during infusion $280
Miltefosine PO 28 days
1.52.5 mg/day
9497% Laboratory strains + Gastrointestinal, nephrotoxicity,
hepatotoxicity, teratogenicity
$70
Paromomycin
sulfate
IM 21 days
15 mg/kg/day
94% (India)
4685% (Africa, depending
on dose)
Laboratory strains + Nephrotoxicity, ototoxicity, hepatotoxicity $10
IV = intravenous administration; IM = intramuscular administration; IL = intralymphatic administration; PO = oral administration.
*
Denitive cure at 6 months.
12 L.H. Freitas-Junior et al. / International Journal for Parasitology: Drugs and Drug Resistance 2 (2012) 1119
Miltefosine (Impavido), also known as hexadecylphosphocho-
line, was simultaneously discovered as an anticancer and antileish-
manial drug (Croft et al., 1987; Scherf et al., 1987). It is the most
recent antileishmanial drug on the market and the rst effective
oral treatment against VL, being recommended as rst line drug
for childhood VL (Bhattacharya et al., 2004). Although its toxicity
is not very high, its teratogenicity is a problem (Sundar, 2007).
The mechanism of action of miltefosine can be a direct action
against the parasite by impairing the lipid metabolism (Croft
et al., 1987) and causing parasite apoptosis (Paris et al., 2004). Mil-
tefosine was also shown to act at the host cell level stimulating the
production of inducible nitric oxide synthetase 2 (iNOS2) that cat-
alyzes the generation of nitric oxide (NO) to kill the parasite within
the macrophage (Wadhone et al., 2009).
A combination therapy of miltefosine with amphotericin B or
paromomycin is very efcient and could be helpful to treat anti-
mony-resistant VL infections in India (Seifert and Croft, 2006).
Paromomycin is an aminoglycoside antibiotic with antileishma-
nial activity. It is used in topical treatment for CL and as an IV drug
for VL (Scott et al., 1992). It is off-patent and has been considered
an orphan drug (Alvar et al., 2006). Paromomycin impairs the mito-
chondrial membrane potential, inhibits protein synthesis, and
leads to respiratory dysfunction. It also alters membrane uidity
and lipid metabolism (Jhingran et al., 2009).
Pentamidine was used as second-line drug in antimony-resistant
VL treatment. High toxicity combined with decreased efcacy in
treatment of patients suggesting resistance (Sundar, 2001), drove
to a complete abandonment of this drug to treat VL in India (Ouel-
lette et al., 2004). However, this compound is still valuable for
combined therapies (Croft and Coombs, 2003). The cellular target
of pentamidine is unknown, but it seems to play a role in the mito-
chondria, as it accumulates in this organelle (Basselin et al., 2000).
Sitamaquine is the second oral drug in development for leish-
maniasis treatment, after miltefosine, and was recently in clinical
trials (Jha et al., 2005). It is an 8-amino-quinoline and preliminary
clinical studies in Kenya and Brazil showed satisfactory efcacy
against different species of Leishmania (Sherwood et al., 1994;
Dietze et al., 2001). A recent study showed that it targets succinate
dehydrogenase causing oxidative stress in L. donovani promastig-
otes (Carvalho et al., 2011).
As indicated, all the main drugs being individually used for the
treatment of leishmaniasis have drawbacks (Table 1). Recent clin-
ical studies have shown the potential benet of antileishmanial
drug combination for VL treatment in India, comparing short-
course multidrug treatment with standard therapy (reviewed by
Olliaro, 2010). The World Health Organization (WHO), following
a meeting in 2010, encourages countries to adopt policies based
on combination therapies when available (WHO, 2010). Neverthe-
less, the lack of adequacy for administration in the eld, toxicity
and resistance issues of the current therapies highlights the need
of new drugs for VL patients.
3. VL drug discovery: new treatments on the way
During the past years several novel drugs or treatments for
tropical diseases have been developed or carried out through drug
repositioning. A sense of urgency to treat these diseases has led to
pragmatic approaches with short and medium-term objectives,
favoring the assessment of combination therapy and repurposing
of known drugs. For example, amphotericin B was primarily devel-
oped as an antimycotic (Oura et al., 1955) and paromomycin is an
antibiotic designed against cryptosporidiosis and amoebiasis
(Courtney et al., 1959). This strategy reduces considerably the time
and cost to have an approved treatment after efcacy conrmation,
with most of the toxicological data and clinical tests already
available (Nwaka and Hudson, 2006). However, this approach is
limited to the number of approved drugs and the probability of
nding a completely new mechanism of action against the parasite
is very low. Given the needs for development of effective and bet-
ter antileishmanials, this strategy is acceptable on the short-term,
but for a mid or long-term solution, a different approach has to be
considered to nd optimal and specic antileishmanials.
Discovery of new drugs for VL treatment should therefore be
seen as a mid or long-term objective. As such and as for any other
drug discovery programs, it is coupled with risk, has to deal with
attrition rate and needs adequate resources, both human and
nancial. This implies the need of optimized drug discovery pro-
cesses, infrastructures, tools and teams, as well as the adequate
funding to be possible to deliver candidates that will be the basis
of the new treatment for VL in the near future.
The target product prole (TPP) is an essential feature of the
drug discovery management tools and plays a central role in the
discovery process, since it depicts the desired outcome of the re-
search and development (R&D) program (Curry and Brown,
2003). With VL being the targeted disease, its specicities, such
as patients living in poor and remote areas with no adequate
health care, imply that the TPP needs to be designed through con-
sultation with expert committees that include stakeholders such as
physicians, local disease control programs, regulators, patient rep-
resentatives, and local and global public health organizations (see
Table 2 for a proposed TPP for VL drug development).
The drug discovery process from the identication of active
compounds (hit or lead) through lead optimization to the clinical
candidate nomination is depicted in Fig. 1. This process is an iter-
ative loop that proceeds through several go/no-go decisions from
the identication of an active compound to a clinical candidate.
Decision gates are usually predened criteria described in the
TPP that a compound should meet in order to be moved forward
along the development chain.
4. Compound screening: rapidly assessing hits
To identify starting points for drug discovery for VL nowadays
two approaches can be undertaken, the molecular target approach
(usually referred to as target-based) and the phenotypic approach
(also called target-free), both having their pros and cons (Gilbert
et al., 2011).
For the target-based discovery approach, the initial step would
be the identication and validation of potential target(s). Biochem-
ical studies or genome mining are some of the available tools to
search for and dene targets. Specically in the case of Leishmania
parasites, the genome sequences of different species are publicly
available (Ivens et al., 2005; Peacock et al., 2007) and a number
Table 2
VL drug target product prole (TPP).
Target label VL and PKDL
Specicity All species
Distribution All areas
Target population Immunocompetent and immunosuppressed
Clinical efcacy >95%
Resistance Active against resistant strains
Safety and tolerability No adverse event requiring monitoring
Contraindications None
Interactions None compatible for combination therapy
Formulation Oral (PO)/intramuscular (IM)
Treatment regimen 1/day for 10 days PO/3 shots over 10 days
*
Stability 3 years in tropical areas
Cost <$10/course
*
This is for primary VL only. PKDL, HIV co-infection and relapse case treatments
may require longer treatment durations.
L.H. Freitas-Junior et al. / International Journal for Parasitology: Drugs and Drug Resistance 2 (2012) 1119 13
of potential targets have been speculated. Some of these targets
were identied and proved to be essential for parasite survival
(Croft and Coombs, 2003), including the parasite dihydrofolate
reductase (DHFR) (Cunningham and Beverley, 2001; Gilbert,
2002), cyclin-dependent kinases (Doerig et al., 2002) and the well
characterized sterol biosynthesis pathway. The latter is a classic
anti-fungal therapeutic target and has been pursued by several lab-
oratories for antileishmanial and antitrypanosomal chemotherapy,
as both Trypanosoma and Leishmania require specic sterols for
growth and cell viability (reviewed by Urbina, 1997; Lepesheva
and Waterman, 2011). Although signicant progress on the identi-
cation and in the validation of these targets have been made, so
far no drug focusing on these targets is being developed for VL.
Recently, efforts were made to select potential parasite targets,
starting with the TDR targets database (https://www.tdrtar-
gets.org). This database was created based on genetic, biochemical
and pharmacological data related to tropical disease pathogens in
addition to computationally predicted druggability (Aguero et al.,
2008). In silico methods applied for the prioritization of these tar-
gets from the TDR database (using criteria such as sequence-de-
rived information, functional data on expression, essentiality,
metabolic pathways and assay development feasibility) ranked
cysteine peptidase C, trypanothione reductase and DHFR as top-
three targets for Leishmania (Crowther et al., 2010).
Another target that has been pursued for antileishmanial drug
discovery is the cyclin-dependent kinase CRK3/CYC6 cyclin com-
plex. Inhibitors of CRK3-CYC6 were discovered on a target-based
high-throughput screening (HTS) recently performed, showing
in vitro activity against promastigotes and intramacrophagic
amastigotes (Walker et al., 2011). Another example of effort target-
ing Leishmania kinases is the Leishdrug Consortium a Framework
Program (FP7, 20072013) created in 2008 for the development
of novel antiparasitic strategies, involving different institutes and
universities from Asia, Europe, Middle-East, South America and
Africa (Dujardin et al., 2010).
In spite of these efforts, the target-based screening approach for
antileishmanial drug discovery stays in the early discovery phase
and has not moved forward to development. Other factors that pre-
clude target-based drug discovery for VL are the low number of
targets well characterized and validated both genetically and
chemically. As for many other elds of discovery, the criteria and
requirements for target validation in VL are loosely dened. Often
it is assumed that protein essentiality per se equals a valid drug
target; however it is crucial to provide evidence of the in vivo role
of the putative target, or at least an essentiality in the intracellular
amastigote form. Due to the particularities of Leishmania parasite
hiding in the parasitophorous vacuole in the macrophage where
the pH is lower than in the surrounding environment, with several
membranes to cross through a pH gradient, and the intricate as-
pects of the host immunity that inuence the development of
leishmaniasis it is not difcult to imagine why an effect on a tar-
get does not translate to an effect on the parasite. Indeed target-
based approaches do not seem to be the most successful strategy
for drug discovery for infectious diseases (Payne et al., 2007).
5. The target-free screening approach
Instead of focusing on a specic enzyme, or other individual
molecule, targeting a pathway might be a promising solution (Hel-
lerstein, 2008). The robustness of critical cellular metabolism relies
on a complex and highly linked adjustable network with redun-
dant or alternate pathways to maintain a usual ow of molecules
and materials in the cell (Fischer and Sauer, 2005). Therefore, the
inhibition of an individual molecule would seldom generate the
expected outcome, a premise in the target-based drug discovery
approach. On the other hand, if a pathway itself can be dened
as the target, it would be a way to select chemical compounds that
are able to interfere in the pathway, no matter the individual path-
way components.
In a more holistic approach, a target-free screening would not
dene any specic molecule or even pathways as a target, but sim-
ply select compounds that are able to eliminate the parasite.
Although viability-based approaches are not applicable to every
case in drug discovery, it is a simple albeit efcient solution for
several infectious diseases including VL. The utmost advantage of
this approach is that in theory a compound can become a drug if
it is potent against the parasite in the disease context without
affecting the host, independently of the target. It has been histori-
cally successful for the discovery of most of the current protozoan
chemotherapy, and also for other elds of discovery and develop-
ment: from 1999 and 2008, for the 75 rst-in-class drugs with
Fig. 1. Hit-to-lead and lead optimization interactive model. Proposed workow for an optimal process to generate a drug candidate against VL.
14 L.H. Freitas-Junior et al. / International Journal for Parasitology: Drugs and Drug Resistance 2 (2012) 1119
new molecular mechanisms of action approved by the Food and
Drug Administration (FDA), 28 were discovered in phenotypic
screenings, 17 were from target-based approaches and the other
30 based on other methodologies (Swinney and Anthony, 2011).
A phenotypic screening is typically a target-free high-content
screening (HCS), a versatile technology collecting information on
several parameters during the screening, enabling thus the
identication and categorization of complex cellular phenomena
(phenotypes). This information is commonly interpreted by auto-
mated software that classies and quanties the cellular compo-
nents and characteristics (Haney et al., 2006; Korn and Krausz,
2007; WHO, 2007; Bickle, 2010). Once a HCS is miniaturized and
adapted to microtiter plate format, the assay can be automated
and become compatible in high-throughput scales.
Besides some activities in academic laboratories, target-free
phenotypic screening efforts to identify compounds active against
Leishmania parasite started under the auspices of the Special Pro-
gram for Research and Training in Tropical Diseases (TDR/WHO).
TDR/WHO accessed compound libraries and tested their activity
in vitro in parasitology laboratories, including the Swiss Tropical
and Public Health Institute (STPH) in Basel, the Laboratory of
Microbiology, Parasitology and Hygiene (LMPH) in Antwerp and
the London School of Hygiene & Tropical Medicine (LSHTM) in Lon-
don. However, these assays were not performed in an automated
fashion and the low-throughput enabled only the screening of a
small number of compounds, reducing thereby the odds of identi-
fying promising series.
Historically the lack of high throughput capacity of phenotypic
screening assays for Leishmania has prevented the access to a crit-
ical mass of quality hits needed for further development consider-
ing the high attrition rate occurring during the drug discovery
process. To address this, numerous attempts to increase the
throughput were made, using either different stages of the para-
site, transgenic parasites, different readouts, different formats, dif-
ferent host cells, but all had their drawbacks (see Table 3) (Singh
and Dube, 2004; Ashutosh et al., 2005; Buckner and Wilson,
2005; Lang et al., 2005; Mandal et al., 2009; Sharlow et al., 2009;
Siqueira-Neto et al., 2010; Osorio et al., 2011). In vitro susceptibil-
ities of L. donovani promastigote and amastigote stages to
antileishmanial reference drugs were also addressed by the scien-
tic community, indicating the practical relevance of stage-specic
differences (Vermeersch et al., 2009; De Muylder et al., 2011). In a
recent study, we found that only 4% of the hits found in a promas-
tigote screening translated into anti-amastigote hits, while about
50% of hits from an intracellular amastigotes screening were con-
rmed to have activity on promastigotes (J Siqueira-Neto and L
Freitas-Junior, unpublished data). Altogether these studies stress
the importance of utilizing intracellular amastigotes as the stage
of study for drug discovery for VL.
Of all the recent efforts to identify new antileishmanials, the
most massive was the HTS of 700,000 compounds by Genomic
Institute of the Novartis Research Foundation (GNF). The assay
used axenic amastigotes in a 1536 well plates format (Bustamante
et al., 2011). Although it is recognized that this form of the parasite
is not fully representative of the intracellular amastigote (Rochette
et al., 2009), some starting points for drug discovery were identi-
ed. The lack of activity translation between axenic amastigote
and intracellular amastigotes has been reported (De Muylder
et al., 2011).
The development of a robust HCS/HTS method using a clinically
relevant form of the parasite intracellular amastigotes infecting
human macrophage was rstly reported in 2010 as a secondary
assay to conrm activity of compounds selected as actives against
the promastigote stage (Siqueira-Neto et al., 2010). In 2011, it was
adapted to a 96 well plate format to screen a few hundred com-
pounds (De Muylder et al., 2011).
The Institut Pasteur Korea (IPK), together with the Drugs for Ne-
glected Diseases initiative (DNDi) developed a HTS/HCS visual
screen in a 384 well plate format in order to nd promising new
chemical compound classes active against Leishmania. The human
macrophage cell line THP-1 (human acute monocytic leukemia)
was used as the host cell and infected with L. donovani metacyclic
promastigotes. The readout of the assay was image-based, and a
customized software was developed for image analysis, allowing
access to information such as infection ratio, number of cells per
well, number of parasites per infected cell, and cell cytoplasm area
among others. This assay was used for screening over 300,000
compounds and more than 350 hits were identied with EC
50
low-
er than 10 lM against intracellular parasites, and no cytotoxicity at
30 lM based on host cell counting (J. Siqueira-Neto and L. Freitas-
Junior, unpublished data). This is a key milestone in the eld of
leishmaniasis drug discovery and is expected to change dramati-
cally the hit generation dynamic for VL.
Another promising approach for the discovery of new antileish-
manial compounds was developed by Peter Melby and collabora-
tors based on ex vivo explant culture system from hamsters
infected with luciferase-transfected L. donovani to screen chemical
compounds for antileishmanial activity (Osorio et al., 2011). The
advantage of this system is that it simulates the complex patho-
physiological environment of the infected spleen in a well plate,
and the compounds are tested in this condition with a diverse host
cell population. While amenable to HTS conditions, this assay re-
quires infected hamsters explants, implying that it is more time
and cost consuming than the assays using THP-1 infected with L.
donovani, thus better suited in secondary screenings for hit priori-
tization and holds promises for lead optimization stages, when
tests of several series of analogues are rapidly needed to advance
potential candidates through the discovery process (see below).
Because of the variety of assays to assess the activity and po-
tency of compounds against the Leishmania parasite in vitro it is
important to have reference assays, standardized and validated
methods to be able to compare data and select good hits and/or
series among the wealth of described active compounds and
screens datasets. After the selection of hit compounds or series of
compounds coming from any screening method, (i) structure
activity relationship studies are performed to prioritize chemical
classes, (ii) derivatives are synthesized, and (iii) the active scaffolds
are identied (lead).
Table 3
Antileishmanial screening formats.
Form of screening Advantages Disadvantages
Transgenic Leishmania Facilitates screening development and permits fast
and sensitive readouts
Requires genetic manipulation of the parasite that may
interfere with biological functions
HTS with promastigotes Easy parasite handling Insect stage of the parasite, poor hit conrmation rate in
amastigote assays
HTS with axenic amastigotes Easy parasite handling Questionable similarity with intracellular amastigotes
HTS with intracellular amastigotes infecting
human macrophage
Most relevant form of the parasite representing the
human disease model
Technically challenging, laborious automation, usually
expensive
Ex vivo splenic explants Simulates pathophysiological infection environment Limited throughput and the use of hamster cells, not human
L.H. Freitas-Junior et al. / International Journal for Parasitology: Drugs and Drug Resistance 2 (2012) 1119 15
6. Hit-to-lead & lead optimization
Major gaps in the drug discovery process for novel antileishma-
nial, once active compounds against Leishmania have been identi-
ed, are twofold. On the one hand, lack of structures adequate
for assessing the potential and identifying possible liabilities of
good hit compounds (hit-to-lead process with synthesis of few
analogues in order to prole hits and assess which can be consid-
ered leads) and the further optimization of leads (lead optimiza-
tion). On the other hand the lack of systematic studies
undertaken to have a proper understanding of the pharmacoki-
netic/pharmacodynamic (PK/PD) relationships for the disease: in
short, how can one relate the exposure of a compound in the body
to its efcacy and safety.
There are rather numerous articles depicting the activity of a
compound against the Leishmania parasite in vitro whatever the
model or a Leishmania target, but very few have been further as-
sessed for in vivo models of the disease. Moreover, the follow-up of
compounds active in vitro and their further optimization through
the synthesis of analogues remains very scarce. Recognizing this
gap, DNDi has put together with partners a lead optimization
structure that includes not only synthetic chemistry capacity but
also PK/PD and efcacy assessment. This integrated process allows
proling active compounds according to specic criteria, having al-
ways in mind the TPP (Chatelain and Ioset, 2011). Once a lead is
introduced into a lead optimization program, it enters a critical
path (Woosley and Cossman, 2007), which promises development
through to the patient access, unless the compound series fails be-
cause of liabilities that cannot be optimized. A proposed criteria for
a lead optimization endpoint and selection of a clinical candidate
for VL treatment is presented in the Table 4. To guarantee rapid
turnaround of data and allow iterative cycles of optimization until
getting to this endpoint, sufcient resources are allocated to the
lead optimization program; in general a team consists of 56
chemists, 23 pharmacologists and dedicated screening facilities
to assess in vitro potency and in vivo efcacy with guaranteed
infrastructures to support medicinal chemistry, in vitro and
in vivo distribution-metabolism-pharmacokinetic (DMPK) and
toxicology.
Indeed, going through rounds of optimization (Fig. 1), looking at
what the body does to the drug (PK) through classical in vitro and
in vivo drug metabolism and pharmacokinetics (DMPK) assays and
what the drug does to the body (pharmacodynamics PD) through
various in vivo animal models, one ends in an optimized lead that
has the desired properties of a potential future drug (see Table 2 for
predened properties of the ideal optimized lead for VL) and is
ready to enter through the classical safety assessment in animals.
To test the in vivo efcacy and potency of the leads and opti-
mized leads, there are different animal models, including mouse,
rat, hamster, dog and non-human primate models. None of these
models, however, completely reproduces the pathology and immu-
nology of the human disease. The in vivo efcacy models for VL
have been shortly reviewed by Gupta and Nishi (2011). Although
dog (Chapman et al., 1979; Keenan et al., 1984) and non-human
primate (Chapman and Hanson, 1981; Chapman et al., 1981; Dube
et al., 1999) models of the disease have been described, their use
remains low and is not really compatible with early stage of drug
discovery as the amount of compound needed for testing in these
models is high.
Rodent models remain, therefore, the standard for drug discov-
ery. Mouse models of leishmaniasis are being extensively used to
get preliminary information of the potential of compounds as they
are fast, reproducible and need relative low amount of compound.
The Balb/c mouse is commonly used for the so-called acute model
and the endpoint of the assay is the parasite load in the liver of the
animals (amastigotes per liver cell nuclei) following 5 days of
treatment (Croft et al., 2006).
According to the classical screening cascade for VL, compounds
for which 80% or more reduction of the liver parasite load in the
mouse model is obtained are then put through more stringent
and relevant chronic hamster model (Melby et al., 2001; Nieto
et al., 2011). The most common strain used is the outbred Syrian
golden hamster (Mesocricetus auratus), dened as a model for
chronic non-cure VL, more similar to the pathology in human with
Table 4
Proposed criteria for clinical candidate selection of a VL drug candidate.
Efcacy Cell-based
a
activity IC
50
6 1 lM
Selectivity 6 50 fold (mammalian isoforms)
Hamster (chronic; liver and spleen): cure after 4 days (PO)
Mouse (acute; liver): cure after 4 days (PO)
Chemistry properties and
prole
Solubility: >0.1 mg/ml (at pH 7.4)
Chirality/bioconversion characterized
COGs amenable to cost effective scale up: <6 steps for synthesis preferred, and <10$/treatment
>2 kg available for GLP studies
DMPK Metabolic stable in S9 liver fraction and liver microsomes of mouse, rat, hamster, dog and
human
Major metabolites identied and characterized qualitatively
No induction and no irreversible inhibition of Cytochrome P450 (5 major isoforms)
Protein binding <85%
Plasma, whole blood and GI uid stability >80% after 4 h.
Oral BA in rats and dogs >30% (in aqueous solutions or clinical accepted formulations)
Elimination half life (oral) rat >120 min
Elimination half life (oral) dog >180 min
Toxicity Evaluation on selected panel of receptors, enzymes and ion channels.
hERG assay IC
50
< 10 lM and ratio of plasma Cmax/hERG IC
20
7 days exploratory toxicology/dose ranging (rats) and identify any organ toxicities: GLP studies
Pharm.chemistry/development API formulation with preliminary stability, suitable for preclinical tests
Human pharmacology/safety Determined starting dose for FIM and projected clinical doses
Preparation of clinical development plan
Intellectual property status Freedom to operate
PO = oral administration; COG = cost of goods; GLP = good laboratory practices; BA = bioavailable; API = active pharmaceutical ingre-
dient; FIM = rst in man; QC = quality control.
a
Cell-based refers to intracellular amastigotes infecting macrophages.
16 L.H. Freitas-Junior et al. / International Journal for Parasitology: Drugs and Drug Resistance 2 (2012) 1119
a more synchronous infection in the liver and spleen (Farrell, 1976;
Gifawesen and Farrell, 1989). The clinic-pathological features of
the hamster model of VL mimic active human disease. Systemic
infection of the hamster with L. donovani results in a relentless in-
crease in visceral parasite burden, progressive cachexia, hepato-
splenomegaly, pancytopenia, hypergammaglobulinemia and
ultimately death (Farrell, 1976). Following 5 days treatment and
at different time points up to 5060 days after end of treatment
parasite burden in spleen, liver and bone marrow is determined.
In this model, AmBisome at 5 mg/kg IV is 100% effective against li-
ver stages but clearance from the spleen and bone marrow was not
achieved (Maes et al., 2004). This model gives us an idea of the tar-
get to achieve to consider the potential of a compound to move for-
ward in development.
In spite of extensive physiopathological characterization, the
two most used models, the mouse and the hamster, have not been
yet characterized as good predictors of compound clinical rele-
vancy for VL, especially considering critical aspects as PK/PD and
drug efcacy in these models. This issue arises, in great part, due
to the neglected nature of VL and the lack thereof of drug discovery
and development in other words, there is no correlation between
clinical and pre-clinical data since there are no pre-clinical candi-
dates being advanced into clinical phases. This situation is unfortu-
nately not exclusive of VL; indeed if one look at the PK/PD issues in
the eld of parasitic infections, with the exception of malaria, very
little is known or undertaken for other diseases (Edwards and
Krishna, 2004). In analogy to antimicrobial research (Drusano,
2004), PK/PD data are critical to better understand the pharmaco-
kinetics of a compound and its effect on the parasite.
For a late stage drug development, the dog model infected with
L. infantum or L. chagasi, should be considered an important model.
First, because in some endemic areas, the dogs are natural reservoir
of the parasite and second, because the infection process and
development is similar to the one occurring in human (Rioux
et al., 1969).
From early discovery by HTS to animal models for pre-clinical
development, several tools are already available for the discovery
and development of a new and more efcient treatment against
VL. The expectations are that promising drug candidates will be
in clinical phase by 2013.
7. Conclusion
Current therapy for leishmaniasis is far from ideal. In recent
years substantial progress has been made in the in search for novel
chemotherapeutics from the initiative of individual academic
labs to consortia and publicprivate partnerships. Cutting-edge
technologies have been combined with optimized assays that en-
abled the screening of a total of near half million compounds for
VL. This and other notorious efforts have generated several poten-
tial starting points for drug development. The downstream pro-
cesses of drug discovery is being established with lead
optimization consortia with the critical mass necessary to a suc-
cessful lead optimization program and the potential delivery of
clinical candidates in the near future. The number of players has
also increased dramatically answering to various incentives.
However, there is still a long way to go. Development of new
technologies together with new models to perform lead optimiza-
tion, as well as information sharing and further partnerships
should pave the way to the identication and development of
new candidates during the following couple of years. It is impor-
tant to highlight that the late discovery process, such as lead gen-
eration and optimization steps, and the drug development process,
with the pre-clinical animal models for drug efcacy and PK/PD,
are still in its infancy for leishmaniasis, given that more systematic
approaches to develop new chemical entities for this important but
neglected disease have started only relatively recently. Therefore
still much about VL chemotherapy is going to be learnt on the
way of development of new drugs to treat leishmaniasis, and the
real measure of success will be the delivery of these future drugs
to the patients in need.
Acknowledgements
The authors would like to thank Dr. Manica Balasegaram and
Ivan Scandale for providing material used in the preparation of this
manuscript. Ideas in this article represent the views of the authors
but not necessarily those of their respective institutions.
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